Neuronal Protection Effects of Blueberries through Inhibition of Key Enzymes involved in the Neurogenerative Diseases

By Chelsea Spitz

This past summer, I was awarded a grant from the Office of Undergraduate Research (OUR) to conduct research in the Chemistry Department under the guidance of Dr. Shuowei Cai. The purpose of this research is to study the neuronal protection effects of blueberries against neurodegenerative diseases and develop the extraction method for blueberries. I also planned on identifying active compounds in blueberries and develop a LC-MS based method to fingerprint the blueberry extract from other extraction methods. Unfortunately, due to the ongoing pandemic, I could not get access to LC-MS system, therefore, my research is mainly focusing on development of extraction method and study the neuronal protection effects of blueberries through inhibition of key enzymes involved in the neurodegenerative disease, including the inhibition kinetics, to understand the mechanism of the neuronal protections of blueberries.

Alzheimer’s disease (AD), the most common form of dementia, is a neurodegenerative disease affecting the structural integrity of the brain. Individuals suffering from AD undergo both steady memory loss and significant cognitive decline as a result of the progressive neuronal damages leading to the death of neurons in brain. AD accounts for 60 to 80 percent of dementia cases, while vascular dementia, due to microscopic bleeding and blood vessel blockage in the brain, is the second most common cause of dementia (Alzheimer’s Association, Alz.org). It is estimated that one in 10 Americans over 65 years of age is currently living with symptomatic AD, and worldwide, 50 million people live with symptomatic AD. AD puts a huge burden on both caregivers and the health system. In 2018, the direct costs to American society for caring of those with AD totaled $277 billion, and it is projected to over $1 trillion by 2050 (Alz.org). Yet, there is no cure for AD and only a handful of drugs have been approved by FDA to manage the symptoms that includes cholinesterase inhibitors and N-methyl-D-aspartate receptor (NMDA) receptor antagonist (i.e. memantine). There is even no treatment that can slow down the progresses of the disease. This may be partly due to the lack of knowledge of the mechanism of AD.

Based on the differences seen in AD’s brains, several potential causes have been hypothesized: deficits in the cholinergic transmission; beta-amyloid plagues (Aβ); tau tangles; oxidative damage and mitochondrial dysfunction; neuronal inflammation; synapse loss; vascular changes; endosomal abnormalities, among others. Several of those hypotheses have been found to be connected to each other, and collectively, they lead to the neuron death. For example, acetylcholinesterase (AChE) is a critical enzyme to regulate the level of the neurotransmitter, acetylcholine. Both Aβ and abnormally hyperphosphorylated tau (p-tau) can increase AChE expression. The increased AChE further influences PS1 and tau-protein kinase GSK-3β. GSk-3β induces hyperphosphorylated tau (P-tau), while PS1 affects the APP processing and Aβ production. Inhibition of AChE not only can rescue the deficit of cholinergic transmissions, but also can potentially reduce Aβ and P-tau.

Tyrosinase is a key enzyme in the biosynthesis of melanin. It catalyzes two reactions: the hydroxylation of tyrosine to L-DOPA and the subsequent oxidation of L-DOPA to dopaquinone. This enzyme may also oxidize dopamine to form melanin pigments through the formation of dopamine quinone, a reaction results in the formation of highly reactive oxygen or nitrogen species (ROS) capable of inducing neuronal cell death. Oxidation stress links to both inflammation and endosomal abnormalities, which hold key for neurodegenerative diseases, including AD.

Our research, therefore, is focusing on examination of neuronal protection effects of blueberries through their inhibition of AChE and tyrosinase. Most phytochemicals are extracted from plants using methanol-based solvent. Residual methanol is highly toxic for human consumption. To explore a safer solvent for extraction of phytochemicals from blueberries, we investigate using ethanol as the extraction solvent. Over the course of the summer, we extract the polar components from blueberries using three different types of solvent system: ethanol alone; methanol alone, and methanol/acetone/water/formic acid (40/40/19/1). Our lab has been used the methanol/acetone/water/formic acid extraction system for extraction of blueberries, and our aim is to compare the biological activities of the blueberry extract using ethanol with those with methanol-based extraction. The activity of AChE was examined using the Ellman method, the tyrosinase activity is determined using L-DOPA as the substrate and monitored the enzymatic product at 490 nm. Both assays were carried out using 96-well microplate, and each sample was run triplicate. To further study the mechanism of inhibition on tyrosinase, we carried out the inhibition kinetics with the blueberry extract from ethanol. The kinetics of tyrosinase was measured every 20 s in 3 min to obtain the initial velocity rate. The contraction of blueberry extract used for inhibition kinetic measurement was 0.25 mg/ml and 0.15 mg/ml.

As shown in Figures 1,2 and 3, the extracts from all three solvent systems showed strong inhibition on AChE and tyrosinase. The extract from methanol/acetone/water/formic acid solvent showed the strongest inhibition on both enzymes. The USDA solvent mixture inhibited the enzymes approximately double that of the other two solvents while the USDA MDS and USDA EDS extracts inhibited relatively close to one another. The methanol-based extract however was still slightly stronger than the ethanol-based extract but overall, they were relatively the same. The IC50s show that the USDA Solv mixture is a much stronger inhibitor than the other two solvents because it requires a lower concentration of the extract to inhibit 50% of the enzyme.

Table 1: IC50 Values For Tyrosinase and AChE

Solvent Mix MDS EDS
Tyrosinase 0.17 mg/mL 1.66 mg/mL 1.14 mg/mL
AChE 0.72 mg/mL 3.48 mg/mL 6.89 mg/mL

 

While blueberry extract from ethanol showed less potent as that from methanol-based solvent, it still showed strong inhibition on two key enzymes that related to neurodegenerative diseases. Here, we demonstrated that ethanol can be a safe alternative to extract the bioactive phytochemicals. We further examined the inhibition kinetics of blueberry extract from ethanol to understand the inhibition mechanism on tyrosinase. As shown in Figure 4, the blueberry extract inhibits tyrosinase in a non-competitive manner (mixed mode inhibition). The inhibition constant Ki and Ki’ are 0.056 mg/ml and 0.82 mg/ml, respectively (Table 2). This suggested that the compounds in blueberry both directly interact with the active site of tyrosinase the substrate-enzyme complex.

Table 2: Inhibition Constant of Blueberry Extract

KI (mg/ml) KI’ (mg/ml)
0.25 mg/ml 0.060 0.079
0.15 mg/ml 0.051 0.085
Average 0.056 (0.006)* 0.082 (0.005)

*: the figure in parenthesis is the standard deviation from the two concentrations of blueberry extract

 

Future Plan:

The plan to continue this project consists of continuing to perform more kinetics assays using USDA MDS extract and to try and see if the data is reproducible. We also plan to work on modeling and studying the structure of the enzyme more closely as well as the compounds found in the extracts from the LC-MS data. Once we gain the access to LC-MS instrument, we will identify the compounds in the ethanol extract, and compare with those from methanol-based extracts. We will further be using NMR to confirm the compounds identified from LC-MS.

The research grant I received from the Office of Undergraduate Research allowed me to learn new skills in the lab such as the enzyme kinetics assays as well as help me find my footing for my research goals. I would like to thank my research advisor Dr. Shuowei Cai for guiding me along the way. As well as the Dean to the College of Arts and Sciences, Dean Entin, and the Office of Undergraduate Research for funding my research this summer.

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